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Methods for the reduction of stutter in microsatellite amplification

a microsatellite and amplification technology, applied in the field of methods for reducing stutter in microsatellite amplification, can solve the problems of complicated analysis of closely spaced microsatellite alleles, and the multiple signal pattern observed for each allele is especially difficult to interpr

Inactive Publication Date: 2002-12-12
APPL BIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0038] (c) detecting alterations of the microsatellite as compared to corresponding microsatellites amplified from control tissue; the amplified microsatellite having a G+C content of greater than 50%; wherein the amplification is performed in the presence of a sufficient amount of sorbitol effective to reduce stutter from that observed in the absence of sorbitol.

Problems solved by technology

However, PCR artifacts exhibited by non-proofreading enzymes and referred to as stutter and the terminal transferase side-reaction can complicate analysis of closely spaced microsatellite alleles.
The multiple signal pattern observed for each allele especially complicates interpretation when two alleles from an individual are close in size (e.g., medical and genetic mapping applications) or when DNA samples contain mixtures from two or more individuals (e.g., forensic applications).

Method used

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  • Methods for the reduction of stutter in microsatellite amplification
  • Methods for the reduction of stutter in microsatellite amplification

Examples

Experimental program
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Effect test

example 1

[0121] PCR reactions were used to amplify the DNA tetranucleotide microsatellite, D2S1338 ((TGCC)(TTCC)) in the presence or absence of sorbitol (FIG. 1). The 50 ul reactions contained 20 mM Tris, 20 mM ammonium sulfate, 4.5 mM magnesium sulfate 1 mM each or dATP, dGTP, dCTP and dTTP, 12 pmoles oligonucleotide primers, 2 ng human genomic DNA template, 5U AmpliTaq Gold.RTM. DNA polymerase in water. In reactions in which sorbitol was added, the reactions contained 2.0 M sorbitol. The reaction conditions were as follows: 95.degree. C. for 11 minutes, followed by 28 cycles of 94.degree. C. for 30 seconds, 55.degree. C. for 4 minutes and 69.degree. C. for 6 minutes, followed by incubation at 60.degree. C. for 45 minutes. Following PCR, samples were mixed with ROX fluorescent size marker (Applied Biosystems). Samples were denatured and separated using a protocol for capillary electrophoresis prepared for an ABI 310 Automated Genetic Analyzer according to the manufacturer's specifications. ...

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Abstract

The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of greater than 50%; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol. The invention also provides compositions containing sorbitol, kits for amplifying microsatellites having a G+C content of greater than 50%, and methods of using all of the foregoing.

Description

[0001] The invention relates to methods, compositions and kits for reducing stutter in polymerase chain reaction amplification of microsatellites. In certain embodiments, the invention relates to the use of sorbitol in polymerase chain reactions in an amount effective to reduce stutter in the amplification of mononucleotide, dinucleotide, trinucleotide, tetranucleotide and pentanucleotide microsatellites.BACKGROUND OF THE RELATED ART[0002] Microsatellites, or short tandem repeats (STRs), consist of tandemly repeated DNA sequence motifs of 1 to 6 nucleotides in length. They are widely dispersed and abundant in the eukaryotic genome, and are often highly polymorphic due to variation in the number of repeat units. This polymorphism renders microsatellites attractive DNA markers for genetic mapping, medical diagnostics and forensic investigation. The combination of PCR and gel or capillary electrophoresis under denaturing conditions has greatly improved the genotyping of microsatellite ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6848C12Q2549/125C12Q2527/137C12Q2521/101
Inventor COTICONE, SULEKHA RAOBLOCH, WILL
Owner APPL BIOSYSTEMS INC
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